OP3‐4 peptide sustained‐release hydrogel inhibits osteoclast formation and promotes vascularization to promote bone regeneration in a rat femoral defect model

Abstract Bone injury caused changes to surrounding tissues, leading to a large number of osteoclasts appeared to clear the damaged bone tissue before bone regeneration. However, overactive osteoclasts will inhibit bone formation. In this study, we prepared methacrylylated gelatin (GelMA)‐based hydrogel to co‐crosslink with OP3‐4 peptide, a receptor activator of NF‐κB ligand (RANKL) binding agent, to achieve the slow release of OP3‐4 peptide to inhibit the activation of osteoclasts, thus preventing the long‐term existence of osteoclasts from affecting bone regeneration, and promoting osteogenic differentiation. Moreover, CXCL9 secreted by osteoblasts will bind to endogenous VEGF and inhibit vascularization, finally hinder bone formation. Thus, anti‐CXCL9 antibodies (A‐CXCL9) were also loaded in the hydrogel to neutralize excess CXCL9. The hydrogel slow released of OP3‐4 cyclic peptide and A‐CXCL9 to simultaneously inhibiting osteoclast activation and promoting vascularization, thereby accelerating the healing of femur defect. Further analysis of osteogenic protein expression and signal pathways showed that the hydrogel may be through activating the AKT‐RUNX2‐ALP pathway and ultimately promote osteogenic differentiation. This dual‐acting hydrogel can effectively prevent nonunion caused by low vascularization and provide long‐term support for the treatment of bone injury.


| INTRODUCTION
Femur fractures and defects caused by surgical resection of osteosarcoma are usually accompanied by two major problems in clinical treatment: nonunion of the fracture 1 and avascular necrosis of the femur head. 2,3 The incidence of femur fractures is concentrated in the elderly, and the treatment of osteosarcoma usually requires the removal of a large amount of cancerous bone tissue, both of two situations are often difficult to treat through autologous bone transplantation. 3 By designing a new type of injectable in situ forming hydrogel to promote bone formation and vascularization, it is expected to replace autologous bone transplantation and become a new treatment method for the treatment of femur injuries. [4][5][6] As a three-dimensional structure network with high water content, hydrogel is beneficial to the delivery of nutrients and the growth of blood vessels. 7,8 In addition, the hydrogel can also be used as a drug carrier to achieve sustained release of drugs or polypeptides. [9][10][11]   polypeptide is a nuclear factor-κB receptor activator ligand (RANKL) binding peptide, which inhibits the formation of osteoclasts by inhibiting the binding of RANKL to RANK on the surface of osteoclasts. [12][13][14] Recent studies have also shown that OP3-4 polypeptide binds to RANK on the surface of osteoblasts, induces membrane receptor aggregation, thereby enhancing osteoblast differentiation. 12,14,15 Therefore, OP3-4 can simultaneously inhibit osteoclast activation and promote osteogenic differentiation during bone tissue regeneration. The slow release of OP3-4 polypeptide will help bone tissue regeneration.
However, in the process of bone tissue regeneration, another problem faced is the vascularization of bone tissue. Insufficient vascularization of bone tissue during the repair process can lead to problems such as bone nonunion. 1 Nevertheless, it is worth noting that the osteoblasts can secrete Chemokine (C-X-C motif) ligand 9 (CXCL9), and CXCL9 will binds to endogenous vascular endothelial growth factor (VEGF) to prevent blood vessel formation. 16,17 Therefore, coordinating osteoblast differentiation and vascularization processes can further accelerate bone tissue healing. The use of anti-CXCL9 antibodies (A-CXCL9) was able to bind excess CXCL9, thereby directly abolishing the effect of CXCL9 on VEGF and improving the vascularization process. 16 Realizing the long-term sustained release of the drug or bioactive factor is the key to ensuring that the implant can play a role in promoting bone formation for a long time. After the polypeptide has been modified by methacrylation, it can be co-cross-linked with the methacryloyl polymer material to fix the polypeptide in the hydrogel to achieve long-term sustained release of the polypeptide. 9 In addition, methacrylated gelatin (GelMA) cannot only combine with polypeptides to achieve sustained release but also promote cell adhesion, proliferation. 18 It also has adjustable mechanical properties to adapt to different tissue applications. 19,20 PCL-PEG-PCL (PCEC) copolymer is a nano micelle composed of medical polymers PCL and PEG. The raw material has extremely high biosafety and PCEC can be used to load drugs or growth factors to achieve long-term sustained release. 21,22 In order to promote bone regeneration while reducing the effect of CXCL9 secreted by osteoblasts on the vascularization process, we designed a GelMA-based hydrogel that sustained-release osteogenesis-promoting polypeptide OP3-4 and anti-CXCL9 antibody embedded in PCEC nanoparticles. In this study, we verified the sustained-release effect of the hydrogel on A-CXCL9, and the inhibitory effect on osteoclasts and the pro-differentiation of rBMSCs into osteoblasts from the biofunctional aspect. Finally, the repair effect of slow-release OP3-4 and A-CXCL9 hydrogel materials on femoral defects was evaluated in a rat femoral defect model, which proved that promoting vascularization can better assist bone regeneration.

| Materials
Gelatin, from cold-water fish skin, was purchased from Sigma-Aldrich

| Synthesis of methacrylylated gelatin
GelMA was synthesized as previously described. 23 Briefly, the solution of gelatin was prepared at a concentration of 8% in a water bath at 60 C. Then, methacrylic anhydride was added dropwise to the gelatin solution at a ratio of 0.6:1 (methacrylic anhydride to gelatin). The reaction was kept under room temperature for 8 h. Finally, the solution was dialyzed against deionized water with cellulose dialysis bag (MwCO = 3500) for 5 days and centrifuged at 8000 rpm for 5 min to remove undissolved impurities. The supernatant after centrifuge was lyophilized at À80 C to obtain the final product GelMA.

| Synthesis of PCEC and A-CXCL9@PCEC nanoparticles
PCEC nanoparticles were obtained following the previous report. 21 A 4 g of PEG and 96 g of anhydrous ε-caprolactone were added to dry three-necked bottle and few drops of tin (II) 2-ethylhexanoate was added to the above solution. The mixture was kept at 130 C for 6 h.
Subsequently, the air in the reaction device was exhausted, and the mixture was heated to 180 C under vacuum and kept for 30 min. The mixture was then cooled to room temperature under the protection of nitrogen and dissolved in dichloromethane, and then the product PCEC was precipitated with excess cold petroleum ether, and finally filtered and dried to obtain a PCEC copolymer.
The anionic PCEC nanoparticles were then derived by the following methods 24

| Preparation of the hydrogel
GelMA, OP3-MA, and A-CXCL9@PCEC were dissolved in PBS buffer according to a certain concentration ratio. LAP was then dissolved in the above mixture at the final concentration of 0.1% (w/v). The composition ratios of different hydrogels are shown in Table 1. The hydrogel was finally obtained by light (405 nm, 3 W/cm 2 ) irradiation for 20 s.
PCEC and A-CXCL9@PCEC nanoparticles were first dispersed in deionized water by ultrasonic, and then added dropwise in copper mesh. The sample was then completely dried at room temperature and observed by TEM. The hydrate diameter and particle size distribution of nanoparticles were observed by dynamic laser scatterometer (DLS, Malvern, Zeta Sizer Nano ZS).

| Chemical structure characterization of GelMA and OP3-MA
The chemical structure of GelMA and OP3-MA was characterized by Fourier transform infrared spectrometer (FTIR, Inova-500M, Varian, USA) and nuclear magnetic resonance spectrometer (NMR, VERTEX 70, Burke, Germany). For FTIR characterization, 5 mg of samples was first mixed with 30 mg of potassium bromide (KBr) and ground into powder, then the powder was pressed into a transparent flake and tested by FTIR. The scan range of the wavenumber was 4000-500 cm À1 , and the resolution was 4 cm À1 . As for NMR characterization, samples were dissolved in deuterated water (D 2 O), and the hydrogen spectrum was detected. Morphology of the hydrogel was characterized by scanning electronic microscope (SEM, S-3400, Hitachi, Japan). Hydrogels were first lyophilized under À80 C to remove water, and then fixed on copper sample stage using carbon conductive tape. The samples were spread with gold for 30 s before observation.

| Characterization of Hydrogel
Compression test of hydrogel was tested by universal testing machine (ELF3200; Bose, USA). Hydrogels were pre-prepared into a cylinder with a height of 6 mm and a diameter of 11 mm. Then samples were compressed using universal testing machine at a stable speed (0.05 mm/s). Compressed length and load were recorded by machine and the strain and stress were calculated by the following formula: where l 0 represents the initial height of sample, while l 1 is the height of sample at different time point. P is the compression load, while A is the cross-sectional area of hydrogel.
Equilibrium-swelling ratio was measured by gravimetric method.
Hydrogels were first lyophilized and weighted to obtain the initial weight (W dry ). Immerse the dried hydrogel to PBS buffer (pH = 7.4) at 37 C, then take them out and weight at selected time point. The Equilibrium-swelling ratio was calculated by the following formula: The degradation behavior with and without lysosome was also measured by gravimetric method. Hydrogels were first lyophilized and weighted to obtain the initial weight (W 0 ). Then the hydrogels were immersed in PBS buffer (pH = 7.4) and 1000 U lysosome PBS solution separately. Finally, the hydrogel was taken out, lyophilized and weighted at selected time point. The weight of hydrogel at different time point was recorded as W t . Degradation ratio was calculated by the following formula: RBMSCs were co-cultured with hydrogel for 1, 3, and 5 days in 5% CO 2 incubator at 37 C. Cells at pre-set time point were washed with PBS buffer for twice and incubated with 10% CCK-8 solution for 1 h. The supernatant of cell-hydrogel co-culture system was collected, and the optical density (OD) value of supernatant at 450 nm was measured by microplate reader. Cell viability was calculated as following:

| Cytoskeleton staining of rBMSCs cocultured with hydrogels
The 500 μl of rBMSCs at cell density of 60,000/ml was seeded on GelMA-based hydrogels and cultured at 37 C for 1, 3, and 7 days.
Hydrogels were washed with PBS and fixed with 4% paraformaldehyde for 10-15 min at selected time. Then 0.5% TritonX-100 was used to treated hydrogels for 5 min. After washed with PBS, TRITC Phalloidin working solution and DAPI were used to stain the cytoskeleton. Finally, the stained images were acquired by confocal laser microscopy.

| Osteogenesis evaluation
After rBMSCs cultured with hydrogels leach liquor for 1, 3, and 5 days, the cells were stained with alkaline phosphatase (ALP) staining kit. After rBMSCs cultured with hydrogels leach liquor for 10 days, the cells were stained with alizarin red staining kit and mineralized nodules were captured by inverted microscope.
Immunofluorescence staining of osteogenic-related proteins was also conducted. After co-cultured with the hydrogel leach liquor, the rBMSCs cell slides were fixed with 4% paraformaldehyde, then the membrane was permeabilized with 0.3% Triton-X, and then blocked with 5% BSA overnight. Then, the cells were incubated with primary antibodies against RUNX2, OCN, AKT and p-AKT at room temperature for 1 h and then incubated with secondary antibody conjugated with fluorescent labels at room temperature for 1 h in dark. Thirty-six female SD rats (200-220 g) were randomly divided into four groups (Blank, GelMA, GelMA/OP3-MA, GelMA/OP3-MA/A-CXCL9@PCEC). Pentobarbital sodium (60 mg/kg) was used for intraperitoneal injection to anesthetize rats. Surgical instruments are preautoclaved. Hair on the right leg was shaved and exposed skin was disinfected by iodophor before surgical. Skin of the right femur was cut longitudinally by scalpel. Then muscle, ligament, and femur were separated by dental scraper to expose the distal side of the femur.

|
Bone defect at the femur epiphysis with a diameter of 2.8 mm and a depth of 3 mm was subsequently created using an electric drill. 25  were conducted through immunohistochemical labeling.

| Statistical analysis
Each group of samples in the experiment contains at least three parallel samples, and the results were shown as average and standard deviation.
The significance was analyzed using Graphpad Prism 7.0. One-way analysis of variance (ANOVA) was performed to evaluate the significance of the experimental data. The statistical significance was *p < 0.05, **p < 0.01, and ***p < 0.001.

| Physicochemical structure characterization of hydrogel and nanomaterials
The hydrogel was obtained by photo-initiated free-radical polymerization between GelMA and OP3-MA, which can sustain release of OP3-4 cyclic peptide 9 to block the activation of NF-κB signaling pathway thus inhibit osteoclast formation and bone resorption. 27 Amphiphilic block copolymers PCEC can spontaneously assemble into nano micelle and carry the anti-CXCL9 antibody (A-CXCL9) 21

| Cell compatibility of hydrogel
The rBMSCs was co-cultured with hydrogels to evaluate the cell compatibility. As shown in Figure 3a, the cell viability increased with the extension of the culture time, showing good cell proliferation. The control group GelMA showed the best effect of promoting cell proliferation, and the cell viability on Days 1, 3, and 5 was 100 %± 6.46%, 174.16% ± 17.61%, and 308.08% ± 2.97%, respectively. This is because gelatin itself contains a large number of short RGD peptides, which can obviously promote cell adhesion and proliferation. 19 Therefore, GelMA hydrogel, and the cell survival rate on Day 5 was 294.08% ± 66.94%, which was not significantly different from GelMA hydrogel. Figure 3b shows the live/dead staining of rBMSCs. Although the rBMSCs showed a low proliferation rate in the GelMA/OP3-MA hydrogel, there were no obvious dead cells during the whole culturing period, indicating that the hydrogel was not cytotoxic, but the addition of OP3-MA and A-CXCL9@PCEC would affect the proliferation of cells. 30 Phalloidin stains the actin in the microfilaments in the cytoskeleton and shows red fluorescence. 31 From the staining of the cytoskeleton in Figure 3c, it could be seen that the rBMSCs cultured on

| In vitro A-CXCL9 release and promoting osteogenesis
A-CXCL9 only binds to PCEC through weak electrostatic interaction and is encapsulated in the hydrogel. With the degradation and diffusion of the hydrogel, A-CXCL9 can be gradually released into the surrounding environment to promote bone formation. Figure S2 showed the standard curve of A-CXCL9 measured by ELISA. Totally 2 μg of A-CXCL-9 was carried in 1 ml of hydrogel. By testing the A-CXCL9 released from the hydrogel, we found that the release of A-CXCL9 reached equilibrium after the 7th day, and the final release amount could reach to 58.78% ( Figure S3).
The in vitro promoting osteogenic differentiation ability of hydrogel was detected by co-cultured with rBMSCs. ALP activity was used to indirectly quantify the ability of rBMSCs to differentiate into osteoblasts. 32 Figure 4a shows that ALP activity all increased for 5 days of indicating that these two hydrogels can better promote bone mineralization and complete the last step of bone formation (Figure 4b).
In order to clarify the relevant mechanism of the above results, we performed immunofluorescence staining on the expression of

| DISCUSSION
The repair of damaged bone tissue involves two important issues: the balance of osteoblasts and osteoclasts 4 and the process of vascularization. 36 However, previous studies have found that CXCL9 secreted by osteoblasts binds endogenous VEGF and prevents VEGF from binding to endothelial cells, thereby interfering with the process of bone vascularization. 16 Therefore, the coordination of the three factors of osteoblasts, osteoclasts, and angiogenesis has become a novel strategy to promote bone regeneration. The OP3-4 polypeptide, as a RANKL-binding peptide, 37,38 can inhibit the activation of osteoclasts 12 while promoting the differentiation of osteoblasts, 15 reconciling the first two therapeutic factors. To reconcile the third factor, the process of vascularization, we designed PCEC nanoparticles to simultaneously release anti-CXCL9 antibody (A-CXCL9) to neutralize excess CXCL9 secreted by osteoblasts. 16,17 Under the constraints of the free diffusion of GelMA hydrogel and the electrostatic interaction of PCEC, the sustained release of A-CXCL9 was close to 60% after 8 days, and the release profile basically reached equilibrium. When the hydrogel was gradually degraded, the remaining A-CXCL9 will be gradually released into the surrounding environment, continuously improving the inhibition of vascularization. 16,17 The entire coordination process was achieved by GelMA-based hydrogels, which have good biocompatibility and degradability and are widely used in the repair of hard tissues such as bone and cartilage. 9 Through in vitro toxicity assessment, we confirmed that the

CONFLICT OF INTERESTS
The authors declare no competing financial interest.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.